Design, Synthesis and Biological Evaluation of Novel MDH Inhibitors Targeting Tumor Microenvironment

MDH1 and MDH2 enzymes play an important role in the survival of lung cancer. In this study, a novel series of dual MDH1/2 inhibitors for lung cancer was rationally designed and synthesized, and their SAR was carefully investigated. Among the tested compounds, compound 50 containing a piperidine ring displayed an improved growth inhibition of A549 and H460 lung cancer cell lines compared with LW1497. Compound 50 reduced the total ATP content in A549 cells in a dose-dependent manner; it also significantly suppressed the accumulation of hypoxia-inducible factor 1-alpha (HIF-1α) and the expression of HIF-1α target genes such as GLUT1 and pyruvate dehydrogenase kinase 1 (PDK1) in a dose-dependent manner. Furthermore, compound 50 inhibited HIF-1α-regulated CD73 expression under hypoxia in A549 lung cancer cells. Collectively, these results indicate that compound 50 may pave the way for the development of next-generation dual MDH1/2 inhibitors to target lung cancer.


Introduction
The rapid growth of cancer cells requires an efficient ATP supply. Cancer cells show alterations in the metabolic pathways that are related to energy production and biosynthetic processes, such as increased uptake of glucose and amino acids and increased breakdown of glutamine and fatty acids. Mutations in EGFR, KRAS, MYC, PI3K, AKT, LKB1, and p53 are known to be involved in cancer metabolism. Targeting energy metabolism has been suggested as an effective strategy for inhibiting cancer cells [1][2][3][4].
To obtain ATP in the electron transport pathway in cells, the cytosolic NADH must be transported into the mitochondria. Because NADH itself cannot be transported into the mitochondria, cells transport reducing equivalents across the mitochondrial membrane using a malate-aspartate shuttle (MAS). The MAS is operated by two pairs of enzymes, malate dehydrogenases (MDH1 and MDH2) and glutamate oxaloacetate transaminases (GOT1 and GOT2), which are localized in the mitochondria and cytoplasm. MDH1 and MDH2 catalyze the reversible conversion of malate to oxaloacetate (OAA) using the NAD/NADH cofactor system [5][6][7][8][9]. Aminooxyacetic acid (AOA), a specific MAS inhibitor, decreases the proliferation of breast adenocarcinoma cells by suppressing GOT1 and GOT2 [10].
The association of MDH1 and MDH2 have been reported in several cancers such as pancreatic and lung cancers. The expression levels of MDH1 and MDH2 are high decreases the proliferation of breast adenocarcinoma cells by suppressing GOT1 and GOT2 [10].
The association of MDH1 and MDH2 have been reported in several cancers such a pancreatic and lung cancers. The expression levels of MDH1 and MDH2 are high in pa tients with lung cancer [4,10]. High expression of MDH1 is significantly correlated wit patient survival, and its knockdown affects cell viability compared with that of MDH [10,11]. A previous report presented LW1497, a dual inhibitor of MDH1 and MDH2, whic showed significant in vivo antitumor effects against colon cancer through the inhibitio of mitochondrial respiration and hypoxia-inducible factor-1 alpha (HIF-1α) accumulatio [12,13]. Another report showed that LW1497 downregulates Slug expression to imped the progression of A549 lung cancer cells by inhibiting epithelial-mesenchymal transitio [14].
In this study, to develop a potent dual inhibitor of MDH1/2 for lung cancer, LW1497 de rivatives were synthesized based on their structure-activity relationships (SARs). Finally, w identified compound 50 as a dual inhibitor of MDH1/2 in lung cancer cells (Figure 1).

Chemistry
The synthesis of the amide compounds 5a-f was completed in four steps, as shown in Scheme 1. The hydroxyl group of phenol 1 was alkylated with methyl propiolate in th presence of triphenylphosphine (PPh3) in toluene, and the resulting (E)-phenoxyacryli methyl ester (2) was saturated with palladium on carbon (Pd/C) under H2 balloon pressur followed by hydrolysis with lithium hydroxide to furnish carboxylic acid 4 at 81% yield [12]. Finally, the T3P-mediated coupling of the resultant carboxylic acid 4 with corre sponding amines yielded a series of amide derivatives 5a-f.

Chemistry
The synthesis of the amide compounds 5a-f was completed in four steps, as shown in Scheme 1. The hydroxyl group of phenol 1 was alkylated with methyl propiolate in the presence of triphenylphosphine (PPh 3 ) in toluene, and the resulting (E)-phenoxyacrylic methyl ester (2) was saturated with palladium on carbon (Pd/C) under H 2 balloon pressure followed by hydrolysis with lithium hydroxide to furnish carboxylic acid 4 at 81% yield [12]. Finally, the T3P-mediated coupling of the resultant carboxylic acid 4 with corresponding amines yielded a series of amide derivatives 5a-f.

Molecular Docking Study for Compound 50
Molecular docking is a vital component of computer-aided drug des range of valuable applications, including the prediction of ligand-target the ranking of compounds based on their docking scores, and the corr scores with potential activity [19,20]. Visualizations of the interactions gen docking studies serves as a guide for optimizing the affinity characteristic ligands. In this study, compound 50 was subjected to an in-depth molecu against both MDH1 and MDH2 to determine its binding patterns. The resu compound 50 displayed a docking score of −4.9 Kcal/mol against MDH attributed to the number of hydrogen bonds formed by compound 50 ag depicted in Figure 2. Compound 50 formed two hydrogen bonds with Gln highlighting the importance of forming a hydrogen bond with this amino activity. The combined structure optimization and SAR studies resulted in th of novel compounds, such as 5d, 5f, 14, 35a, 44a, 50, 56, 57a, and 57b, which inhibition on the activities of both MDH1 and MDH2. However, compoun and 57d showed MDH1 activity, whereas 5b and 5c only showed MDH2

Molecular Docking Study for Compound 50
Molecular docking is a vital component of computer-aided drug des range of valuable applications, including the prediction of ligand-target the ranking of compounds based on their docking scores, and the corr scores with potential activity [19,20]. Visualizations of the interactions gen docking studies serves as a guide for optimizing the affinity characteristic ligands. In this study, compound 50 was subjected to an in-depth molecu against both MDH1 and MDH2 to determine its binding patterns. The resu compound 50 displayed a docking score of −4.9 Kcal/mol against MDH attributed to the number of hydrogen bonds formed by compound 50 ag depicted in Figure 2. Compound 50 formed two hydrogen bonds with Gln highlighting the importance of forming a hydrogen bond with this amino activity. The combined structure optimization and SAR studies resulted in th of novel compounds, such as 5d, 5f, 14, 35a, 44a, 50, 56, 57a, and 57b, which inhibition on the activities of both MDH1 and MDH2. However, compound and 57d showed MDH1 activity, whereas 5b and 5c only showed MDH2 a
For the SAR exploration of portion D, we chose to start with compound 5d by keeping the -OCF 3 group intact at the meta position on the amide phenyl ring in portion A and (4-(3,3-dimethylbutyl) alkyl chain in portion B. Replacement of the flexible etherlinked aliphatic chain from 5d with an amine-linked piperidine ring (50) exhibited dual inhibitory activity against MDH1 and MDH2 (IC 50 = 3.33 ± 0.18 and 2.24 ± 0.09 µM, respectively). Compound 51, which contains a piperazine group, showed decreased dual inhibitory activity (Table 5). Replacing the amine-linked piperidine with a more flexible amine-linked aliphatic chain (56) led to an increase in the activity against MDH1 (IC 50 = 1.79 ± 0.04 µM) but also maintained similar inhibitory activity against MDH2. When the hydrogen atom in 56 was substituted with aliphatic groups, such as methyl (57a), isopropyl (57b), and cyclobutyl (57c), it retained similar potency against both MDH1 and resulted in a two-to three-fold improvement in the inhibitory activity against MDH2. Interestingly, the N-acylated derivative (57d) was more selective for MDH1 than for MDH2 (IC 50 = 1.41 ± 0.47 and >5 µM, respectively) ( Table 6).

Molecular Docking Study for Compound 50
Molecular docking is a vital component of computer-aided drug design as it offers a range of valuable applications, including the prediction of ligand-target binding modes, the ranking of compounds based on their docking scores, and the correlation of these scores with potential activity [19,20]. Visualizations of the interactions generated through docking studies serves as a guide for optimizing the affinity characteristics of the studied ligands. In this study, compound 50 was subjected to an in-depth molecular examination against both MDH1 and MDH2 to determine its binding patterns. The results showed that compound 50 displayed a docking score of −4.9 Kcal/mol against MDH1, which can be attributed to the number of hydrogen bonds formed by compound 50 against MDH1, as depicted in Figure 2. Compound 50 formed two hydrogen bonds with Gln14 and Asn130, highlighting the importance of forming a hydrogen bond with this amino acid residue for activity.
The molecular docking study of compound 50 against MDH2 revealed a docking score of approximately −5.9 Kcal/mol. Compound 50 formed a hydrogen bond with the Gly35 residue within the binding site and two additional hydrogen bonds with the Ile36 and Lys105 residues, as shown in Figure 3.

ATP Production in the Presence of MDH Inhibitor
Malate transported to the interior of the mitochondria is oxidized to OAA by mitochondrial MDH2, thereby generating NADH, which then enters the ETC to produce ATP. Because inhibition of MAS is expected to reduce ATP production in cancer cells, the total amount of ATP was measured after treatment with compound 50. Cells treated with compound 50 showed reduced ATP level in A549 cells ( Figure 4A). In addition, an increase in ADP/ATP was observed, indicating the inhibition of ATP synthesis by oxidative phosphorylation ( Figure 4B). The molecular docking study of compound 50 against MDH2 revealed a dock score of approximately −5.9 Kcal/mol. Compound 50 formed a hydrogen bond with Gly35 residue within the binding site and two additional hydrogen bonds with the Il and Lys105 residues, as shown in Figure 3. Favorable interactions are color-coded as follows: green-hydrogen bonds, light blue-halogen interaction, orange-π-charge, dark pink-π-π stacking interactions, purple-π-sigma interactions, light pink-hydrophobic interactions. Residues shown in light green form weak van der Waals interactions.
We then examined whether the ATP production pattern was altered by drug treatment using an XF analyzer for real-time metabolic analysis ( Figure 5). As expected, the total ATP decreased by 14.6% and 28.6% in cells treated with 5 µM and 10 µM of compound 50, respectively ( Figure 5).  Favorable interactions are color coded as follows: green-hydrogen bonds, light blue-halogen interaction, orange-π-charge, dark pink-π-π stacking interactions, purple-π-sigma interactions, light pink-hydrophobic interactions. Residues shown in light green form weak van der Waals interactions.

ATP Production in the Presence of MDH Inhibitor
Malate transported to the interior of the mitochondria is oxidized to OAA by mitochondrial MDH2, thereby generating NADH, which then enters the ETC to produce ATP. Because inhibition of MAS is expected to reduce ATP production in cancer cells, the total Favorable interactions are color coded as follows: green-hydrogen bonds, light blue-halogen interaction, orange-π-charge, dark pink-π-π stacking interactions, purple-π-sigma interactions, light pink-hydrophobic interactions. Residues shown in light green form weak van der Waals interactions.  We then examined whether the ATP production pattern was altered by drug treatment using an XF analyzer for real-time metabolic analysis ( Figure 5). As expected, the total ATP decreased by 14.6% and 28.6% in cells treated with 5 μM and 10 μM of com- nation of ADP/ATP ratio; * p ≤ 0.05 and ** p ≤ 0.01, compared with the control.
We then examined whether the ATP production pattern was altered by drug treatment using an XF analyzer for real-time metabolic analysis ( Figure 5). As expected, the total ATP decreased by 14.6% and 28.6% in cells treated with 5 μM and 10 μM of compound 50, respectively ( Figure 5).

Compound 50 Inhibits Hypoxia-Induced Accumulation of HIF-1α
Previously, it has been reported that the inhibition of MDH1 or MDH2 enzymes is related to HIF-1a expression levels [12]. We investigated whether compound 50 decreases HIF-1α accumulation in A549 cells ( Figure 6). Compound 50 decreased HIF-1α accumulation in a dose-dependent manner ( Figure 6). Additionally, we confirmed that compound 50 suppressed the protein expression of HIF-1α target genes, such as GLUT1 and PDK1 (Figure 6), as clearly shown by the Western blot assay.

Compound 50 Inhibits Hypoxia-Induced Accumulation of HIF-1α
Previously, it has been reported that the inhibition of MDH1 or MDH2 enzymes is related to HIF-1a expression levels [12]. We investigated whether compound 50 decreases HIF-1α accumulation in A549 cells ( Figure 6). Compound 50 decreased HIF-1α accumulation in a dose-dependent manner ( Figure 6). Additionally, we confirmed that compound 50 suppressed the protein expression of HIF-1α target genes, such as GLUT1 and PDK1 (Figure 6), as clearly shown by the Western blot assay.

Compound 50 Suppresses the Expression of CD73 Regulated by HIF-1α
It has been reported that CD73 expression is induced by HIF-1α under hypoxic conditions [21]. Therefore, we examined whether the expression of CD73 is inhibited by compound 50 under hypoxia in A549 lung cancer cells. As shown, CD73 expression increased by HIF-1α was significantly decreased by compound 50 in a concentration-dependent manner. To confirm the effect of compound 50 on CD73 expression, we performed ELISA for CD73. Compound 50 reduced CD73 levels in A549 cells in a dose-dependent manner (Figure 7). These results confirmed that compound 50 showed a significant inhibition of hypoxia-induced transcription and CD73 activity.

Compound 50 Suppresses the Expression of CD73 Regulated by HIF-1α
It has been reported that CD73 expression is induced by HIF-1α under hypoxic conditions [21]. Therefore, we examined whether the expression of CD73 is inhibited by compound 50 under hypoxia in A549 lung cancer cells. As shown, CD73 expression increased by HIF-1α was significantly decreased by compound 50 in a concentration-dependent manner. To confirm the effect of compound 50 on CD73 expression, we performed ELISA for CD73. Compound 50 reduced CD73 levels in A549 cells in a dose-dependent manner (Figure 7). These results confirmed that compound 50 showed a significant inhibition of hypoxia-induced transcription and CD73 activity.
ditions [21]. Therefore, we examined whether the expression of CD73 is inh pound 50 under hypoxia in A549 lung cancer cells. As shown, CD73 expres by HIF-1α was significantly decreased by compound 50 in a concentrat manner. To confirm the effect of compound 50 on CD73 expression, we per for CD73. Compound 50 reduced CD73 levels in A549 cells in a dose-depe (Figure 7). These results confirmed that compound 50 showed a significan hypoxia-induced transcription and CD73 activity.

Discussion
The reprogramming of the core metabolism in tumors confers selecti vantages, such as the ability to enhance cell proliferation and promote tum progression [22][23][24][25][26][27]. One of the mechanisms to suppress tumor growth is t of the cancer cell's metabolism. MAS plays a crucial role in the net transfer produced in the cytoplasm into the mitochondria. The transportation of cy as an electron donor into the mitochondria is a major purpose of MAS i

Discussion
The reprogramming of the core metabolism in tumors confers selective growth advantages, such as the ability to enhance cell proliferation and promote tumor growth and progression [22][23][24][25][26][27]. One of the mechanisms to suppress tumor growth is the impairment of the cancer cell's metabolism. MAS plays a crucial role in the net transfer of the NADH produced in the cytoplasm into the mitochondria. The transportation of cytosolic NADH as an electron donor into the mitochondria is a major purpose of MAS in cancer cells. Therefore, MDH1 and MDH2, which are components of MAS, are the key molecules involved in ATP production via the process of mitochondrial oxidative phosphorylation [7,28,29]. In this study, we synthesized compound 50, a potent MDH1/MDH2 dual inhibitor that suppressed MAS function in A549 lung cancer cells.
First, we measured ATP production to elucidate the inhibitory effects of compound 50 on mitochondrial respiration. Total ATP production was significantly reduced in A549 cells treated with compound 50, which increased the ADP/ATP ratio. These results suggest that compound 50 inhibits the growth of A549 cells by inhibiting energy metabolism. In all cancer types, ATP supply is solely dependent on glycolysis and OXPHOS. Therefore, the selective inhibition of prevalent tumor energy metabolism may have a significant impact on cancer treatment. However, it can be argued that such a treatment may also severely affect the host's energy metabolism.
Cancer cells significantly depend on cytosolic NADH production from glucose, fatty acids, and glutamine for ATP production [30]. Elevated glycolysis in cancer cells has been proposed as a mechanism that accelerates oxidative phosphorylation. MAS exerts control over NADH/NAD + homeostasis to maintain the activity of mitochondrial lactate dehydrogenase and enables the aerobic oxidation of glycolytic L-lactate in the mitochondria.
In contrast, high aerobic glycolysis distinguishes cancer cells from normal cells and has been exploited to detect tumors in vivo. Lactate consumption in cells treated with compound 50 did not change significantly. Cancer cells consume the secreted lactate to produce ATP through the TCA cycle and oxidative phosphorylation processes [31,32]. Some cancer cells use lactate as a substrate for TCA intermediates via monocarboxylate transporters (MCT1/4) as well as for ATP production [32]. Lactate can be converted to pyruvate using lactate dehydrogenase (LDH) and can be further converted to acetyl-CoA by ATP-citrate lyase for fatty acid synthesis.
Under hypoxia, HIF-1α increases the transcriptional expression of various genes that are involved in cancer progression, metastasis, angiogenesis, and resistance to therapy [33]. We found that compound 50 decreased the expression of HIF-1α and its target genes, such as glucose transporters (GLUT1 and GLUT3) and PDK1, preventing pyruvate entry into the TCA cycle [5,6]. We also observed a decrease in CD73 expression in the HIF-dependent adenosinergic pathway, which impairs NK cell function in tumors. Recently, the development of agents that can either block CD73 and/or target HIFs concurrently with NK cell-based therapies has emerged as an immunotherapeutic strategy with significant potential for the treatment of solid tumors.

General Procedures
The commercial chemicals and solvents used were of reagent grade. All reactions were performed under a nitrogen atmosphere in oven-dried glassware. Reactions were monitored using thin-layer chromatography on 0.25-milimeter silica plates (E. Merck; silica gel 60 F254). The products were purified using flash column chromatography (Biotage) using silica gel 60 (230-400-mesh Kieselgel 60). Proton nuclear magnetic resonance (NMR) spectra were recorded on a Varian 400 MHz spectrometer (Varian Medical Systems, Inc., Palo Alto, CA, USA). The chemical shifts are provided in δ values (ppm), and the coupling constants are in hertz (Hz). 13 C NMR spectra were recorded on the Varian 100 MHz spectrometer. The melting points were measured using the Thermo Scientific 9200 melting point apparatus. The mass spectra were recorded using high-resolution mass spectrometry (HRMS) (electron ionization MS) on a Waters G2 QTOF mass spectrometer. The purity of the final products was determined using reversed-phase high-performance liquid chromatography (RP-HPLC). The Waters Corp. HPLC system employed a YMC C18 (HS12S05-1564WT) column (5 µM, 12 nm) that had the dimensions of 4.6 mm × 150 mm and was equipped with an ultraviolet (UV) detector set at 254 nm. The RP-HPLC mobile phases used were (A) water with 0.05% trifluoroacetic acid (TFA) and (B) acetonitrile. The purity of the compounds was assessed using a gradient of 25% B to 100% B in 35 min. The purity of all biologically evaluated compounds was >95%.

General Procedure for Acid-Amine Coupling
To a solution of acid (1.0 equiv), amine (1.1 equiv), and N, N-diisopropylethylamine (DIPEA; 3.0 equiv) in tetrahydrofuran (THF; 25 mL), we added 50% propylphosphonic anhydride (T3P) in ethyl acetate (EtOAc; 2.0 equiv). The resulting mixture was stirred at room temperature for 12 h. The reaction mixture was diluted with water (20 mL), and the aqueous phase was extracted using EtOAc (3 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (15-45% EtOAc in hexane).

General Procedure for the Michael Addition of Phenol with Acrylates
To a solution of phenol (1.0 equiv) in methyl or ethyl acrylate (20 mL), we added 4-dimethylaminopyridine (DMAP; 1.0 equiv) at room temperature. The resulting mixture was stirred at 100 • C in a microwave for 2 h. The reaction was quenched using saturated sodium bicarbonate solution (20 mL), and the aqueous phase was extracted using EtOAc (3 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0-15% EtOAc in hexane).

General Procedure for Ester Hydrolysis
To a solution of methyl or ethyl ester (1.0 equiv) in THF:H 2 O (10:1), we added lithium hydroxide (LiOH; 2.0 equiv). The resulting mixture was stirred at room temperature for 12 h. The reaction mixture was evaporated under reduced pressure, and the crude residue was redissolved in water (20 mL) and acidified with 3 N hydrochloric acid (HCl; pH 6). The resulting precipitate was collected via suction filtration and dried under vacuum.

General Procedure for Suzuki Coupling
To a solution of halogen compound (1.0 equiv) and corresponding boronic acid (1.2 equiv) in 1,4-dioxane: H 2 O (9:1), we added cesium carbonate (Cs 2 CO 3 ; 3.0 equiv). The resulting mixture was degassed with nitrogen for 5 min. Tetrakis(triphenylphosphine) palladium (Pd(PPh 3 ) 4 ; 0.05 equiv) was added to the degassed solution, and the mixture was heated at 100 • C for 12 h in a sealed tube. The reaction mixture was cooled to room temperature and diluted with water (20 mL), and the aqueous phase was extracted using ethyl acetate (3 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The crude residue was purified using silica gel column chromatography (10-15% EtOAc in hexane).

General Procedure for Reductive Amination
To a solution of amine (1 equiv) in dichloromethane (DCM; 30 mL), we added aldehyde and/or ketone (1.2 equiv) and acetic acid (0.1 mL). The mixture was stirred at room temperature for 2 h, and sodium cyanoborohydride (NaCNBH 3 ; 1.0 equiv.) was then added to the stirring solution. The resulting mixture was stirred at room temperature for 12 h. The reaction was quenched with aqueous sodium bicarbonate solution (30 mL), and the aqueous phase was extracted using DCM (3 × 40 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified using silica gel column chromatography (0-25% EtOAc in hexane). The spectral data for the final compounds is available in the supplementary information (Figures S5-S98).

Enzyme-Linked Immunosorbent Assay (ELISA)
The prepared samples were incubated with the same amount of antibody cocktail for 1 h at room temperature and then washed three times with PT buffer. After washing, the plates were developed by adding 100 µL of TMB development solution, and the reaction was stopped by adding 100 µL of stop solution. The amount of CD73 was quantitatively measured according to the instructions on the Human CD73 Simplestep ELISA kit (Abcam, Cambridge, UK).

Ligand Setting
The chemical structure preparation procedure was performed using ChemDraw software. Ligands were prepared by sketching the 2D structure of each ligand, and 3D structures of all ligands were produced using a molecular mechanical (MM) geometry optimization approach. The Epik function in the LigPrep module of Maestro was used in accordance with the Hammett and Taft methods to return the pKa values and 3D structure files for multiple tautomers and ionization states that are likely to exist under the specified conditions. This was accomplished by setting the physiological pH (pH 7.4) at the protonation state step and the geometry by repeating the optimization step. Accordingly, Epik can be automatically used by LigPrep to enumerate the tautomers and protonation states.

Receptor Preparation
The crystal structures of MDH1 (PDB:5MDH, resolution 2.4 Å) and MDH2 (PDB:4WLO, resolution 2.5 Å) were retrieved from the Protein Data Bank server [35,36]. The protein structures were subjected to the protein preparation procedure using Schrodinger software 2020, which included: (i) removing all water molecules from the crystal structure (implicit solvation was used); (ii) assignment of bond orders; (iii) adding hydrogen atoms; (iv) setting the physiological pH (pH 7) via the protonation states of the corresponding amino acid residues using PROPKA software 20 in the Schrodinger molecular modeling package; and (v) restraining the minimization of added hydrogen atoms. The active site used in the docking study was constructed from chain A of the crystal structure.

Conclusions
In this study, various structural modifications were employed for the hit compound LW1497 to design new scaffolds. Accordingly, a series of novel (2,4,4-trimethylpentan-2yl)benzene derivatives were synthesized and screened for their inhibitory effect against MDH1 and MDH2 enzymes. Optimization of the SAR study resulted in the identification of a series of novel dual or selective MDH1 and MDH2 inhibitors. Compounds 5d and 35a showed significant inhibitory activity against both MDH1 and MDH2, whereas compounds 5f, 14, 44a, 50, 56, 57a, and 57b showed similar activity against both enzymes, compared with the positive control LW1497. Compounds 39b, 39c, 39d, and 57d displayed selectivity towards MDH1, whereas 5b and 5c showed selective MDH2 activity. In addition, the growth inhibition results of all tested compounds revealed that compound 50, which contains a piperidine linker, showed a two-fold improved potency in A549 (IC 50 = 3.94 ± 0.04 µM) and H460 (IC 50 = 3.67 ± 0.06 µM) cell lines compared with the hit compound LW1497. The most potent compound 50 decreased ATP production by inhibiting the MAS shuttle. As expected, compound 50 suppressed the expression of HIF-1α target genes as well as hypoxia-induced HIF-1α accumulation. Therefore, compound 50, now LW2393, may serve as a promising lead for the development of novel MDH1/2 inhibitors for the treatment of lung cancer.